Aluminum Phosphate Vaccine Adjuvant: Analysis of Composition and Size Using Off-Line and In-Line Tools

Abstract

Purpose: Aluminum-based adjuvants including aluminum phosphate (AlPO₄) are commonly used in many human vaccines to enhance immune response. The interaction between the antigen and adjuvant, including the physical adsorption of antigen, may play a role in vaccine immunogenicity and is a useful marker of vaccine product quality and consistency. Thus, it is important to study the physicochemical properties of AlPO₄, such as particle size and chemical composition. Control of the vaccine adjuvant throughout the manufacturing process, including raw materials and the intermediate and final product stages, can be effectively achieved through monitoring of such key product attributes to help ensure product quality.

Methods: This study focuses on the compositional analysis of AlPO₄ adjuvant at the intermediate and final manufacturing stages using the off-line methods Fourier-Transform Infrared (FTIR) and Raman spectroscopy, X-ray Photoelectron Spectroscopy (XPS), and the in-line method Attenuated Total Reflectance (ATR). Particle size distribution of AlPO₄ was measured off-line using Laser diffraction (LD) and in-line using Focused Beam Reflectance Measurement (FBRM®).

Results: There was no observable difference in size distribution between the intermediate and final stage AlPO₄ by off-line and in-line analysis, in both small- or large-scale production samples. Consistent peak shifts were observed in off-line and in-line infrared (IR) spectroscopy as well as off-line XPS for both small- and large-scale AlPO₄ manufacturing runs. Additionally, IR spectroscopy and FBRM® for size distribution were used as in-line process analytical technology (PAT) to monitor reaction progress in real-time during small-scale AlPO₄ manufacturing from raw materials. The small-scale adsorption process of a model protein antigen (Tetanus toxoid) to AlPO₄ adjuvant was also monitored by in-line ReactIR probe.

Conclusion: This study demonstrated that in-line PAT can be used to monitor particle size and chemical composition for the various stages of adjuvant manufacturing from raw materials through intermediate to final adjuvant product stage. Similar approaches can be utilized to help assess lot-to-lot consistency during adjuvant manufacturing and vaccine product development. Moreover, the use of in-line PAT is highly conductive to advanced manufacturing strategies such as real-time product release testing and automated processes of the future.

Keywords: Aluminum phosphate adjuvant (AlPO4); Focused beam reflectance measurement (FBRM®); Fourier transform infrared spectroscopy (FTIR); Laser diffraction (LD); Particle size distribution; Process analytical technology (PAT); Raman spectroscopy; X-ray photoelectron spectroscopy (XPS).

Graphical abstract

Fig. 1

(a) Particle size distribution profiles of intermediate (green trace) and final (red trace) AlPO₄ measured by LD; (b) FTIR and (c) Raman spectra of intermediate (green trace) and final (red trace) AlPO₄ manufactured at large scale.

Fig. 2

High resolution XPS spectra collected with the 15° take-off angle for Al 2p, P 2p, and O 1 s of intermediate (blue trace) and final (black trace) AlPO₄ sample. Smooth curves represent the fitting. The horizontal lines on the graph represent the baseline used for fitting.

Fig. 3

(a) AlPO₄ precipitation reaction monitoring in real-time (b) Overlay of four AlPO₄ intermediate lots size distribution profiles by LD (orange, dotted) and FBRM® (blue, solid); each lot was prepared by mixing AlCl₃ and Na₃PO₄ raw materials in the EasyMax reactor (c) The size distribution of manufacturing scale AlPO₄ by LD, intermediate and final (dotted), and by FBRM®, intermediate and final (solid).

Fig. 4

(a) The inline IR spectral overlay of AlCl₃ (red trace) and Na₃PO₄ (blue trace); (b) Inline IR monitoring of AlPO₄ adjuvant formation during the small-scale precipitation reaction. The normalized IR peak height corresponding to AlCl₃, AlPO₄ transient intermediate are represented by the solid gray, solid blue, and solid red traces respectively. The dotted green trace represents the volume of Na₃PO₄ (in mL) added.

Fig. 5

IR Spectra of (a) intermediate and (b) final AlPO₄ (small-scale) using inline analysis; (c) IR Spectra of intermediate (red, magenta, and purple with a broad plateau) and final (green, blue, and pink with a narrow plateau) stage AlPO₄ (large-scale) using inline analysis.

Fig. 5

IR Spectra of (a) intermediate and (b) final AlPO₄ (small-scale) using inline analysis; (c) IR Spectra of intermediate (red, magenta, and purple with a broad plateau) and final (green, blue, and pink with a narrow plateau) stage AlPO₄ (large-scale) using inline analysis.

Fig. 6

IR spectral overlay during inline monitoring of Tetanus Toxoid adsorption to final AlPO₄ adjuvant. Red trace represents Tetanus Toxoid IR spectrum at the beginning of adsorption reaction. Amide II peak decreases during adsorption (downward arrow), while Amide I peak becomes more prominent (upward arrow), which is consistent with FTIR spectrum of Tetanus Toxoid reported previously [5].